The present invention provides modified avidin and streptavidin compounds that have optimal blood clearance kinetics for use in a two-step approach to deliver a molecule to a target site. In particular, to hasten streptavidin""s blood clearance, carbohydrate moieties are covalently bonded to streptavidin. To delay streptavidin""s blood clearance, esterified carbohydrate moieties are covalently bonded to streptavidin. To prolong avidin""s blood clearance, avidin is deglycosylated and/or neutralized by alkylation of its lysine amino acids. The present invention further provides targeting agents, such as monoclonal antibodies, conjugated to modified streptavidin and avidin. In a two-step method of imaging or therapy, biotin conjugates are used to deliver radionuclides, cytotoxic drugs, magnetic resonance imaging agents, fluorochromes and other agents suitable for imaging and therapy to target-bound conjugates of modified streptavidin or avidin and antibodies or other targeting agents. The present invention is also directed to the sugar containing intermediates used to prepare the streptavidin modified compounds of the present invention.
Various diagnostic, therapeutic, fluorescent and enzyme linked applications utilize cell or tissue specific targeting agents as delivery systems for radioactive, paramagnetic, cytotoxic or therapeutic agents. Any agent which is specific for a lesion or site of interest can potentially act as a targeting agent. For example, polyclonal and monoclonal antibodies can be produced which exhibit considerable specificity for certain cell or tissue types. Many other agents, including toxins such as diphtheria toxin, exhibit cell specificity and can be used to deliver diagnostic or therapeutic agents. The technique of delivery of monoclonal antibodies (MAbs) has been investigated for cancer therapy as well as for diagnosis of cancer, thromboembolism and cardiac myopathy.
For successful imaging with directly labeled antibodies, sufficient labeled MAb must localize at the target site to provide enough signal for detection. Target-to-background ratios must be high in order to achieve adequate contrast between target-bound radioactivity and background levels in other organs, tissues and blood. A major obstacle to successful imaging with directly labeled antibodies is the high background activity of free circulating radiolabeled MAbs due to prolonged circulation and accumulation in liver, kidney and spleen, the normal metabolic sites for Abs. Furthermore, the toxic effects of high radiation doses must be considered in both radioimmunotherapy and radioimmunoimaging. Such obstacles are also a consideration for methods utilizing targeting agents other than monoclonal antibodies.
Galactose protein modification has been utilized in an attempt to manipulate clearance of antibodies. For example, Ong et al. (1991) Cancer Res. 51:1619 and Mattes (1987) JNCI 79:855 have conjugated galactose to radiolabeled MAbs to increase the blood clearance rate of the MAbs in both diagnostic and therapeutic techniques. U.S. Pat. No. 4,401,647 discloses the galactose modification of albumin for liver imaging. Vera et al. (1985) J. Nucl. Med. 26:1157 and Stadalnik et al. (1992) Investig. Radiol. 28:64 have conjugated galactose to albumin for functional imaging studies of the liver. Both of these procedures result in radionuclide accumulation in the liver. This is problematic for imaging liver or chest lesions. In addition, liver metabolism increases, which creates problems for imaging and therapy due to accumulation of radioactivity in radiosensitive organs and tissues, especially bone marrow.
To overcome such obstacles, xe2x80x9cpre-targetingxe2x80x9d approaches have been investigated. See, e.g., Hnatowich, et al (1987) J. Nucl. Med. 28: 1294. In the conventional one-step method the radionuclide is linked to the MAb either directly or via a bifunctional chelating agent. In the pre-targeting approach the antibody is unlabeled, but conjugated to a binding moiety such as avidin or streptavidin. Unlabeled antibody is administered, and antibody which does not localize to the target site is allowed to clear from circulation or removed by a clearing agent before the administration of radioactivity. The radioactivity is then administered in a chemical form which has high affinity for the antibody, e.g., bound or chelated to the binding partner of the moiety conjugated to the antibody.
To provide the diagnostic or imaging agent in a form with high affinity for the antibody, two-step methods have been designed to exploit the high affinity of avidin and streptavidin for biotin. Avidin, a 67 kilodalton (kD) glycoprotein found in egg whites, has an exceptionally high binding affinity (Kd=10xe2x88x9215M) for biotin. Avidin consists of four subunits, each capable of binding one biotin molecule. Streptavidin, a similar protein produced in Streptomyces avidinii, shares significant conformation and amino acid composition with avidin, as well as high affinity and stability for biotin. However, streptavidin is not glycosylated and reportedly exhibits less non-specific binding to tissues. Streptavidin is widely used in place of avidin because of its lower non-specific binding. Biotin, a member of the B-complex vitamins, is essential for amino acid and odd-chain fatty acid degradation, gluconeogenesis and fatty acid synthesis and is normally found in the enzyme bound form as biocytin.
In the prior art approach to radioimaging or radiotherapy, antibodies are coupled with either biotin or streptavidin and administered to the subject, followed by administration of radiolabeled streptavidin or biotin, respectively. Hnatowich et al. (1987); Kalofonos et al. (1990) J. Nucl. Med. 31:1791; Paganelli et al. (1992) Eur. J. Nucl. Med. 19:322; Yao et al (1995) J. Nucl. Med. 36:83. A three-step approach, which involves the administration of biotinylated antibody, followed by streptavidin and then radiolabeled biotin, has also been investigated. Paganelli et al. (1988) Int. J. Cancer 2:121. These multistep procedures generally require large doses of protein, and long time durations, often days, to complete. In particular, the prolonged circulation of streptavidin-conjugated antibodies requires either a significant time interval to allow clearing or the use of a clearing agent before administration of radiolabeled streptavidin or biotin. Paganelli et al. (1992) Eur. J. Nucl. Med. 19:322; U.S. Pat. No. 4,863,713 to Goodwin et al. Conversely, avidin conjugated moieties would be expected to clear too quickly, and would likely not be useful in pre-targeting approaches due to unacceptably low target accumulation.
Accordingly, the pre-targeting method of the prior art is a complicated system that suffers from practical limitations, including, for example, the pharmacokinetics of avidin and streptavidin. Streptavidin and avidin exhibit markedly different pharmacokinetics after intravenous injection, with avidin clearing from the blood much faster than streptavidin. Thus, for a rapid two step procedure, streptavidin-containing moieties clear slowly, necessitating a delay in the injection of radiobiotin until blood levels have decreased. Conversely avidin clears too quickly and accumulates in the liver and kidney, thus resulting in low target accumulation. Avidin""s rapid blood clearance results from its inherent positive charge and its mannose terminal sugars which probably bind to mannose receptors present in the liver on Kupffer cells.
Thus, the directly labeled antibody approaches of the prior art suffer from background dosimetry problems, and the pretargeting approaches of the prior art are complicated, requiring large doses, multiple steps, and significant amounts of time to perform.
The present invention overcomes the deficiencies of the prior art. In particular, the present invention provides modified avidin and streptavidin compounds that have optimal blood clearance kinetics for use in a rapid method for delivery of an agent to a target site. Further, the avidin and streptavidin compounds of the invention are not radiolabeled, thus avoiding interference from background activity. Because of the optimal clearance kinetics and rapid hepatocyte internalization of the present compounds, any of the subsequently administered radiolabeled biotin that does not become target bound is quickly filtered by the kidney and removed from the body. The present invention thus satisfies the prior art need for a rapid and efficient method of imaging and therapy.